Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes

Abstract

The yeast Sfp1 protein regulates both cell division and growth but how it coordinates these processes is poorly understood. We demonstrate that Sfp1 directly controls genes required for ribosome production and many other growth-promoting processes. Remarkably, the complete set of Sfp1 target genes is revealed only by a combination of ChIP (chromatin immunoprecipitation) and ChEC (chromatin endogenous cleavage) methods, which uncover two promoter binding modes, one requiring a cofactor and the other a DNA-recognition motif. Glucose-regulated Sfp1 binding at cell cycle "START" genes suggests that Sfp1 controls cell size by coordinating expression of genes implicated in mass accumulation and cell division.

Keywords: G1/S regulon; START; Sfp1; cell cycle; cell growth; chromatin endogenous cleavage (ChEC); ribosomal protein gene; ribosome biogenesis; transcription.

Figure 1.

Regulation of growth-related genes by Sfp1. (A) Scatter plot comparing Rpb1 ChIP-seq signal (log₁₀ normalized read counts) in SFP1 (left panel) and pGAL1-SFP1 (right panel) strains grown in 2% raffinose (x-axis) or 1 h following 2% galactose addition (y-axis). RP genes (green); RiBi genes (red); all other genes (gray). (B) Scatter plots comparing Rpb1 ChIP-seq fold-change (log₂) relative to t = 5 (left) and 20 (right) min following rapamycin addition in a Sfp1 anchor-away strain (y-axes) to Rpb1 ChIP-seq change 60 min following galactose addition in a pGAL-SFP1 strain (x-axes). (C) Direct comparison of time points in B focusing on Sfp1-repressed (left) and Sfp1-activated (right) genes. (D) Average TBP binding profiles, centered on TATA or TATA-like element (Rhee and Pugh 2012), at 20 min following vehicle or rapamycin addition to a Sfp1 anchor-away strain at the indicated gene groups. In the top panel, average values for RiBi genes are depicted in solid lines, those for RiBi-like genes in dashed lines.

Figure 2.

Glucose-regulated binding of Sfp1 at G1/S (START) regulon genes. (A) Genome browser tracks comparing Sfp1-TAP ChIP-seq signals in glucose or galactose medium, or in an untagged strain grown in glucose, at the genomic loci indicated below. (B) Heat map (right) showing ratio of Sfp1-TAP ChIP-seq signal from glucose-grown versus galactose-grown cells. Positions of genes whose promoters are bound by Swi4 or Ifh1 are indicated in orange or green, respectively. (C) Scatter plots comparing Sfp1-TAP ChIP-seq signal in the indicated strains and growth conditions (RAF, raffinose; GAL, galactose; GLU, glucose). For the pGAL1-SFP1-TAP strain ChIP-seq was performed 60 min following galactose.

Figure 3.

ChIP and ChEC detect distinct classes of Sfp1 promoter binding sites. (A) Genome browser tracks comparing Sfp1-TAP or untagged ChIP-seq signals (yellow background) to Sfp1-MNase and free MNase ChEC-seq signals (blue background), the latter at the indicated time points following calcium addition. The position and direction of transcription of individual RiBi (top) and RP (bottom) genes are shown below. Control strains (ctrl) lack either the TAP or MNase tags. (B) Heat maps showing Sfp1-TAP ChIP-seq under endogenous expression (WT) or after pGAL1-SFP1 overexpression (OE); Sfp1 ChEC-seq signal after 30 or 150 sec of Ca⁺² treatment, or Ifh1 ChEC-seq signal after 150 sec of Ca⁺² treatment at the indicated categories of genes (right). Signals for a window of −400 to +100 bp relative to the +1 nucleosome (0) are displayed (x-axis). Control for ChIP (untagged strain) and ChEC (free-MNase, 20 min following Ca⁺² addition) are also shown.

Figure 4.

Cofactor-dependent and DNA sequence motif-dependent modes of Sfp1 binding. (A) Sfp1 occupancy (qPCR-ChIP) at the indicated promoters and times following auxin treatment in an Ifh1-AID strain; fold enrichment relative to ACT1 was normalized to values at t = 0, which were set to 1. (B) Sfp1 occupancy as in (A) following rapamycin treatment in a Swi4 anchor-away strain. (C) Average plots of Sfp1-MNase cleavage centered on the indicated motifs at 30 or 150 sec after Ca⁺² activation of MNase. Control averages (free-MNase cleavage 20 min after Ca⁺²) are also shown.

Figure 5.

Schematic representation of Sfp1 binding and regulation. See text for details.